Journal of Lipid Research, Vol 26, 745-754, Copyright © 1985 by Lipid Research, Inc.
Modification of human high density lipoprotein (HDL3) with tetranitromethane and the effect on its binding to isolated rat liver plasma membranes
GK Chacko
Apolipoprotein E-free high density lipoproteins (HDL) bind to various cells
and cell membrane preparations, with properties typical of ligand- receptor
interactions. In order to further characterize the binding sites and to
investigate the functional role of binding, a chemically modified HDL
without the specific binding properties would be highly desirable. We have
reacted human HDL3 with tetranitromethane, a relatively specific nitrating
reagent for tyrosine residues, in 50 mM Tris HCL buffer, pH 8.0, and at a
reagent concentration 10 times the molar excess of tyrosine residues. The
resulting nitrated HDL3 completely lost its ability to bind to high
affinity saturable binding sites of rat liver plasma membranes, as
determined by competitive binding with 125I-labeled HDL3, and also by
direct binding assays using 125I-labeled nitrated HDL3. Although nitrated
HDL3 did not bind to the high affinity saturable binding sites, it bound to
the membranes, but the binding was not saturable, and was not competed for
by unlabeled nitrated HDL3. On agarose gel electrophoresis, pH 8.6, the
nitrated HDL3 moved ahead of the control HDL3, indicating an increase in
negative charges in the molecule. No difference in size was noted in the
nitrated HDL3 when analyzed either by negative stain electron microscopy or
by gel filtration chromatography. Spectroscopic analysis of the nitrated
HDL3 at pH 8.0 revealed a prominent absorption with maximum at around 360
nm, but none in the region expected for nitrotyrosine residues. At pH 10.0,
however, the nitrated HDL3 showed an absorption band with a maximum at
around 440 nm, possibly related to nitrotyrosine residues. Nitrotyrosine
was detected in the nitrated HDL3 on amino acid analysis. Comparison of the
amino acid analysis of the nitrated HDL3 and control HDL3 showed no
difference in composition of any of the amino acids except tyrosine;
tyrosine content was reduced more than 90% in the nitrated HDL3.
SDS-polyacrylamide gel electrophoresis analysis of apoproteins of nitrated
HDL3 revealed changes in apolipoprotein profile. Bands corresponding to the
apolipoproteins of the starting HDL3 almost disappeared and a series of new
bands appeared at the high molecular weight region of the gel, indicating
extensive cross-linking of apolipoproteins during the reaction. In
addition, a substantial amount of phospholipids and cholesteryl esters, but
not unesterified cholesterol, was found covalently linked, possibly through
the unsaturated centers of the fatty acid chains, to
apolipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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